Study On The Mechanism Of Modified Shenqi Dihuang Decoction In Improving Diabetic β-cell Dysfunction

BackgroundDysfunction of β cells mainly refers to the impaired cell-induced insulin secretion function or the reduced cell numbers under the pathological factors of glycolipid toxicity,which is central to the pathogenesis of type 2 diabetes.Apoptosis is the ultimate form of the reduced βcell numbers and dysfunction.A previous literature review found that there is a very complex interaction between autophagy and β-cell apoptosis.On the one hand,moderate autophagy enables target cells to maintain cellular homeostasis and protect them from apoptosis by removing damaged organelles when they are exposed to stress.On the other hand,excessive activation of autophagy can promote apoptosis and cause cellular damage.Therefore,autophagy and apoptosis are closely related to β-cell dysfunction,but the research and prevention means for autophagy and apoptosis of pancreatic β-cells are still very limited.Shenqi Dihuang Decoction comes from Shenshi Zusheng Book.Professor Ni Qing improved the original formula by replacing ginseng with codonopsis,rehmannia with raw dioscorea,and adding dioscorea,zedoary and fenugreek.The all-around attacking and tonicizing effect,supporting the righteousness and eliminating the wickedness,has the effect of Qi-Yin dual tonicity,nourishing the kidney and strengthening the spleen.The results of network pharmacology showed that this remedy may have regulatory effects on PI3K,AKT and other proteins.Therefore,this study is intended to investigate the effect of modified Shenqi Dihuang Decoction on the improvement of β-cell dysfunction in combination with in vivo and external experiments,and to further explore the molecular mechanism of the PI3K/AKT/mTOR signaling pathway-based regulation of β-cell autophagy and apoptosis.ObjectiveCombined with in vitro and in vivo experiments,we observed the effects of modified Shenqi Dihuang Decoction on improving high-fat combined with low-dose STZ-induced T2DM rats,and high sugar combined with sodium palmitate-induced RINm5F cell injury,and further explored the mechanism of action of modified Shenqi Dihuang Decoction on improving pancreatic β-cell dysfunction based on PI3K/AKT/mTOR signaling pathway from the perspective of cell autophagy and apoptosis.Methods1.In vivo part:75 SPF male SD rats were randomly fed with ordinary diet.The other 65 rats were fed with high-fat diet for 5 weeks and intraperitoneal injection of STZ(35mg/kg)to establish T2DM rat model.T2DM rats with successful modeling were randomly divided into normal group,model group,high-dose modified Shenqi Dihuang Decoction group,medium dose modified Shenqi Dihuang Decoction group,low-dose modified Shenqi Dihuang Decoction group and metformin group.Each group was gavaged with corresponding drugs for 8 weeks.We recorded the general state of rats in each group and the changes of body weight,food intake and drinking water intake every week before and after administration,and detected the fasting blood glucose(FBG)before administration(0 week),4 weeks and 8 weeks after administration.At the end of 8 weeks,we performed glucose tolerance test(OGTT)and evaluated the glucose metabolism of rats in each group.The rats in each group were killed after 8 weeks of intervention,and the blood was taken from the abdominal aorta for the subsequent detection of glycosylated albumin(GA),fasting insulin(FINS),blood lipids(CHO,TG,LDLC,VLDL,HDL-C),so as to evaluate the islet function and lipid metabolism of rats in each group;The indexes of liver and pancreas were measured,and the pancreatic tissue was stained with HE.Based on the above test results,evaluate the role of modified Shenqi Dihuang Decoction in improving glucose and lipid metabolism,improving pancreatic tissue morphology and reducing pancreatic tissue injury in T2DM rats,so as to improve islet cell dysfunction and maintain glucose homeostasis.To further clarify the mechanism of action of modified Shenqi Dihuang Decoction in enhancing β-cell dysfunction,we used transmission electron microscopy to observe autophagic vesicles,protein immunoblotting to detect the expression levels of PI3K/AKT/mTOR signaling pathway-related proteins AKT,p-AKT,mTOR,p-mTOR,and their downstream autophagic effector molecules LC3-Ⅱ and Beclin-1,and RT-PCR to detect the expression levels of Caspase-3,Bax,and Fas mRNA.2.In vitro part:25 mM high glucose combined with 250 μM sodium palmitate induced the injury model of RINm5f cells for 24 hours.SD rats were gavaged with high dose of modified Shenqi Dihuang Decoction determined in animal experiment and animal drinking water to prepare modified Shenqi Dihuang decoction containing serum and blank serum for standby.CCK-8 method was used to evaluate the cytotoxicity of modified Shenqi Dihuang decoction containing serum on cells,and to determine the high,medium and low concentration range of modified Shenqi Dihuang decoction containing serum intervention.Then,RINm5f cells were divided into blank control group,model group,high serum containing modified Shenqi Dihuang Decoction,medium serum containing modified Shenqi Dihuang Decoction and low dose serum containing modified Shenqi Dihuang Decoction,and the morphological changes of RINm5f cells in each group were observed by inverted phase contrast microscope;The level of insulin secretion in each group of cells was detected by radioimmunoassay,and the apoptosis of each group of cells was detected by flow cytometry and Hoechst 33342 kit to clarify the effect of modified Shenqi Dihuang Decoction in reducing apoptosis and improving pancreaticβ-cell dysfunction.To further clarify whether it is based on the PI3K/AKT/mTOR pathway to regulate autophagy to exert islet cytoprotective effects,the autophagy inducer rapamycin and the autophagy inhibitor 3MA were used to verify their mechanism of action,and the autophagic vesicles in each group were observed by transmission electron microscopy;the expression levels of PI3K/AKT/mTOR signaling pathway-related proteins AKT,p-AKT,mTOR,p-mTOR,and their downstream autophagy effector molecules LC3-II and Beclin-1 were detected by protein immunoblotting;and the expression levels of Caspase-3,Bax,Fas,and Bcl-2 mRNA were detected by RT-PCR to validate the in vivo experimental results from the cellular level.Results1.In vivo part:(1)Modified Shenqi Dihuang Decoction can significantly improve the symptoms of overeating in T2DM rats(P<0.05),but it has no significant effect on body weight and drinking symptoms(P>0.05);(2)Modified Shenqi Dihuang Decoction could significantly reduce FBG and GA in T2DM rats(P<0.05),improve glucose tolerance(P<0.05),increase fasting insulin secretion(P<0.05),and improve HOMA-β and ISI;(3)Modified Shenqi Dihuang Decoction could reduce the liver index(P<0.05),reduce the levels of TG,LDL-C and VLDL(P<0.05),increase HDL-C(P<0.05)and improve lipid metabolism in T2DM rats;(4)HE staining showed that modified Shenqi Dihuang Decoction could improve the pancreatic tissue morphology,reduce the vacuolar degeneration of islet cells and reduce the injury of pancreatic tissue in T2DM rats.Among the above pharmacodynamic experimental results,the high dose group of modified Shenqi Dihuang decoction has the best effect.(5)Transmission electron microscopy showed that the number of autophagic vesicles increased in the pancreatic tissue of the model group,and mitochondrial autophagy and mitochondrial changes in the shape of "fluffy balls" could be observed.Compared with the model group,the number of mitochondrial autophagy and autophagic vesicles decreased in the modified Shenqi Dihuang decoction group,and the typical double-layer membrane-like structure was occasionally seen,and the mitochondrial morphological disorder was improved compared with that of the model group;(6)The results of Western blot showed that the low,medium and high dose groups of modified Shenqi Dihuang Decoction could significantly up regulate the expression of p-Akt and p-mTOR protein,and down regulate the expression levels of LC3-Ⅱ and beclin-1 protein(P<0.05);(7)The results of RT-PCR showed that the expression of Caspase-3 and Bax mRNA could be significantly down regulated in the medium and high dose groups of modified Shenqi Dihuang Decoction(P<0.05),and the expression of Fas mRNA could be significantly down regulated in the low,medium and high dose groups of modified Shenqi Dihuang Decoction.2.In vitro part:(1)The induction of islet RINm5F cell injury by 25 mM high sugar combined with 250 μM sodium palmitate can successfully establish a stable in vitro model of islet cell injury.(2)The serum containing modified Shenqi Dihuang Decoction in the range of 20%has no toxic effect on cells.The appropriate concentration range of modified Shenqi Dihuang Decoction in the intervention of RINm5f cell injury model is 5%-15%;(3)The serum containing modified Shenqi Dihuang Decoction could significantly increase the activity of RINm5f cells(P<0.05),improve cell morphology and promote the level of insulin secretion(P<0.05);(4)Hoechst 33342 staining showed that modified Shenqi Dihuang Decoction could improve the nuclear morphology and reduce the number of dense stained nuclei and cell fragments;The results of flow cytometry showed that the drug containing serum of modified Shenqi Dihuang Decoction could significantly reduce the apoptosis rate of RINm5f cell injury model(P<0.05),and the effect of 15%drug containing serum was the most significant.Therefore,15%modified Shenqi Dihuang decoction containing serum was used to study the follow-up molecular mechanism.(5)The results of transmission electron microscope showed that a large number of autophagy bodies could be observed under the microscope of the model group,and typical autophagy bodies could also be seen near the nucleus,that is,the closed and spherical structure formed by wrapping part of the cytoplasm by double-layer membrane or single-layer membrane;autophagy bodies were occasionally seen in 15%modified Shenqi Dihuang Decoction group,which was less than that in the model group;(6)The results of Western blot showed that 15%modified Shenqi Dihuang decoction containing serum could significantly up regulate the expression of p-Akt and p-mTOR protein(P<0.05),and down regulate the expression of LC3-Ⅱ and beclin-1 protein(P<0.05);(7)RT-PCR showed that 15%modified Shenqi Dihuang Decoction group could significantly down regulate the mRNA expression of Caspase-3,Bax and Fas(P<0.05),and up regulate the mRNA expression level of Bcl-2(P<0.05).After the addition of rapamycin,the effect was reduced or offset.Conclusion1.Modified Shenqi Dihuang Decoction can significantly regulate glucose and lipid metabolism,improve the morphological structure of pancreas,promote insulin secretion and protect the function of islet cells in T2DM rats.2.Modified Shenqi Dihuang Decoction can significantly improve the vitality of RINm5f cells,improve cell morphology,promote insulin secretion and inhibit the apoptosis of RINm5f cells.3.The effect of modified Shenqi Dihuang Decoction in improving pancreatic islet β-cell dysfunction may be achieved by activating PI3K/AKT/mTOR signaling pathway,downregulating the expression of downstream autophagy effector molecules LC3-Ⅱ and Beclinl protein,regulating pancreatic islet cell β-cell autophagy,and down-regulating Caspase-3,Bax,and Fas mRNA expression to inhibit cell apoptosis.